Detection of nucleic acids in a sample is useful in diagnostic, therapeutic, forensic, agricultural, food science applications and other areas. Methods of nucleic acid detection generally include those that use physical separation of a nucleic acid, such as by capturing the nucleic acid in or on a matrix or support followed by detecting the presence of captured nucleic acids and/or amplification products made therefrom. Detection can take place by a number of methods known in the art, such as mass spectrometry, sequencing, a dye or intercalating agent, or by hybridizing a detectable probe to the nucleic acid. Some methods indirectly detect nucleic acids by producing a product made from using a target nucleic acid as a template and detecting the product, e.g., detecting an RNA transcript made from a DNA, or a translated protein made from an RNA transcript. Other indirect methods detect a product made by an enzymatic reaction associated with the nucleic acid to be detected, e.g., an enzyme-linked probe hybridized to the target nucleic acid which produces a detectable response when the enzyme's substrate is provided. Some methods of nucleic acid detection rely on amplifying a nucleic acid sequence to produce a larger quantity of nucleic acid that is detected. Examples of amplification methods include producing many copies of a cloned sequence and in vitro amplification procedures that use enzymatic synthesis of multiple copies of a nucleic acid sequence.
Many of the techniques for detecting nucleic acids require the presence of a relatively large amount or proportion of the target nucleic acid in the sample, while other techniques use nucleic acid amplification to increase the amount or proportion of the nucleic acid to be detected from a smaller amount of the target nucleic acid in a sample. Enrichment of some or all of the nucleic acid present in a sample may facilitate detection of the nucleic acid of interest. Many known procedures for nucleic acid enrichment and detection are laborious, time-consuming, or require use of equipment or hazardous chemicals (e.g., chaotropes, mutagens, or radioactive compounds) that make such procedures undesirable for many applications, such as for rapid screening of many samples, point-of-care diagnostics, or detection at a site outside of a laboratory. Thus, there remains a need for a method that provides relatively simple procedures and sufficient sensitivity and/or specificity to detect a nucleic acid of interest.
The physical nature or relative abundance of some nucleic acids may impede their detection in a sample. For example, small RNA (about 17-27 nt), such as microRNA (miRNA), small or short interfering RNA (siRNA), short hairpin RNA (shRNA), and small nuclear RNA (snRNA) are difficult to separate from other sample components and/or to detect by using known methods. Small RNA are often relatively rare in a biological sample, which contributes to the difficulty of their detection. Further complicating the detection of these small RNA molecules is that these short template RNA species often share high sequence homology between closely related family members. Most small RNA detection methods in the art use general RNA isolation techniques, like Invitrogen Trizol reagent, Stratagene Total RNA, ABI mirVana and ABI Total RNA, (Invitrogen, Carlsbad, Calif.; Stratagene, La Jolla, Calif.; ABI, Foster City Calif.), all of which provide an indiscriminant abundance of RNA species that may include the target small RNA molecule(s) and include many other non-target nucleic acids. These non-target nucleic acids often interfere with analysis of target sequence. These one size fits all approaches introduce bias and experimental error into small RNA expression profiling experiments. Because small RNA are regulatory molecules that modulate or silence gene expression via RNA interference (RNAi), they are important molecules for understanding biological processes and disease states and as disease preventive or therapeutic agents. Studies have shown that differential small RNA expression occurs in cancerous and non-cancerous cells. Thus, there is a need for compositions and methods that rapidly and accurately isolate small RNA from a sample for downstream analysis. There is a need to rapidly and accurately isolate and detect the presence of one or more small RNA in biological samples to determine their abundance, relative abundance, expression level, stability, therapeutic efficacy, or other characteristics in a biological sample. There is a further need to rapidly and accurately detect relative amounts of differentially expressed small RNA in a variety of biological samples as an indicator of a cancerous condition and/or as a part of a cancer prognosis by determining metastatic potential of a tumor and in developing a suitable treatment thereof.
Current methods for detecting small RNA in biological samples are time consuming and imprecise. Common techniques include PCR (RT and qRT), in situ hybridization, nuclease protection assays, Northern blots to detect RNA, Western blots, immunoassays, and fluorescence detection assays (PCT App. Nos. WO 00/44914, Li et al., WO 05/04794, Bumcrot et al.). Detection methods also exist wherein an additional sequences is added to the small RNA to facilitate priming and detection, such as adding a single universal extension primer or adapter to every miRNA in a sample (Chen et al. U.S. Pat. No. 7,601,495; Raymond et al. RNA, 11:1737-44 (2005)). These added nucleic acid sequences are then used as primer binding sites for amplification of the small RNA and/or as capture probe-binding sites, (Mullinax et al., US Pat. Pub. No. 2008-0182235; Jacobsen et al. WO 05/098029). In addition, these added nucleic acid sequences can be used as primers for a first amplification (Chen et al. Nuc. Acid. Res., 33(20):e179 (2005)). Another method for amplifying and detecting a small RNA includes using a bridging oligo that is complementary to both a small RNA molecules and a unique nucleic acid molecule followed by a ligating step to join the small RNA and the unique nucleic acid (Yeakley, US Pat. Pub. No. 2006-0019258). These methods, unfortunately, are inaccurate; particularly as throughput increases so too does the variability in result data (e.g., Mestdagh, Chen et al. Nuc. Acid. Res. 36(21):e143 (2008) and Nelson et al., Biochim. Biophys Acta, 1179(11):758-65 (2008)). Thus, the methods in the art are not fully sufficient for providing reliable small RNA expression data for use in diagnosing disorders in which one or more of these small RNAs play a role. Moreover, sample-processing methods in the art are crude and inadequate for sample-to-answer automation, for identifying high throughput processing of a large number of specimens, particularly for biomarker validation and diagnostic applications. There is a need for a method for accurately detecting the presence and relative abundance of one or more small RNAs in a sample for diagnosing and monitoring a disorder, as well as for prognosing the disorder and/or monitoring the efficacy of a treatment of the disorder. There is also a need for methods and reagents useful with high throughput analysis of and diagnostics using biomarkers, such as small RNA.
This application responds to the need for rapid, accurate and efficient nucleic acid detection assays by disclosing methods and compositions useful for the isolation and detection of one or more nucleic acids in samples, including one or more small RNA in biological samples, and that are amenable with fully automated platforms.